PKCα regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes

Phospholipases A2 (PLA2) are potent regulators of the inflammatory response. We have observed that Group IV cPLA2 activity is required for the production of superoxide anion (O2−) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404–2411.]. We have previously identified PKCα as a kinase pathway required for monocyte O2− production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508–17515.]. We therefore investigated the potential interaction between PKCα and cPLA2 by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA2 enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA2 activity. To distinguish between PKCα and PKCβ isoenzymes in regulating cPLA2 protein phosphorylation and enzymatic activity, we employed our previously characterized PKCα or PKCβ isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764–3771]. Suppression of PKCα expression, but not PKCβ expression, inhibited cPLA2 protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA2 phosphorylation and activation. We also found that cPLA2 co-immunoprecipitated with PKCα and vice versa. In vitro studies demonstrated that PKCα could directly phosphorylate cPLA2.and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O2− in monocytes defective in either PKCα or cPLA2 expression. Taken together, our data suggest that PKCα, but not PKCβ, is the predominant cPKC isoenzyme required for cPLA2 protein phosphorylation and maximal induction of cPLA2 enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCα and cPLA2 in O2− generation are solely due to their seminal role in generating arachidonic acid.